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Abstract:

The present invention relates to methods of preventing airway remodeling
using A2B adenosine receptor antagonists. This invention finds
utility in the treatment and prevention of asthma, COPD, pulmonary
fibrosis, emphysema, and other pulmonary diseases. The invention also
relates to pharmaceutical compositions for use in the method.

Claims:

1. A method for the treatment and prevention of airway remodeling and/or
pulmonary inflammation by administration of a therapeutically effective
amount of an A2B receptor antagonist to a mammal that is genetically
and/or environmentally predisposed to airway remodeling and/or pulmonary
inflammation.

2. The method of claim 1 wherein the method is for the treatment and
prevention of airway remodeling.

3. The method of claim 1, wherein the method is for the treatment and
prevention of pulmonary inflammation.

4. The method of claim 1, wherein the mammal is human.

5. The method of claim 1, wherein the administration is systemic.

6. The method of claim 5, wherein the administration is oral.

7. The method of claim 5, wherein the administration is intravenous.

8. The method of claim 5, wherein the administration is intramuscular.

9. The method of claim 5, wherein the administration is intraperitoneal.

10. The method of claim 1, wherein the A2B receptor agonist is
administered by inhalation.

11. The method of claim 1, wherein the mammal suffers from a disease state
chosen from asthma, pulmonary fibrosis, and COPD.

12. The method of claim 11, wherein the disease state is asthma.

13. The method of claim 11, wherein the disease state is pulmonary
fibrosis.

14. The method of claim 11, wherein the disease state is COPD.

15. The method of claim 1, wherein the A2B receptor antagonist has
the structure of Formula I or Formula II: ##STR00029## wherein:R1
and R2 are independently chosen from hydrogen, optionally
substituted alkyl, or a group -D-E, in which D is a covalent bond or
alkylene, and E is optionally substituted alkoxy, optionally substituted
cycloalkyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted heterocyclyl, optionally substituted
alkenyl, or optionally substituted alkynyl, with the proviso that when D
is a covalent bond E cannot be alkoxy;R3 is hydrogen, optionally
substituted alkyl or optionally substituted cycloalkyl;X is optionally
substituted arylene or heteroarylene;Y is a covalent bond or alkylene in
which one carbon atom can be optionally replaced by --O--, --S--, or
--NH--, and is optionally substituted by hydroxy, alkoxy, optionally
substituted amino, or --COR, in which R is hydroxy, alkoxy or amino;with
the proviso that when the optional substitution is hydroxy or amino said
substitution cannot be present on a carbon atom adjacent to a heteroatom;
andZ is hydrogen, optionally substituted monocyclic aryl or optionally
substituted monocyclic heteroaryl;with the proviso that(a) Z is hydrogen
only when Y is a covalent bond and X is optionally substituted
1,4-pyrazolene attached to the purine ring by a carbon atom; and,(b) when
X is optionally substituted arylene, Z is an optionally substituted
monocyclic heteroaryl other than optionally substituted imidazole.

16. The method of claim 15, wherein:R1 and R2 are independently
hydrogen, optionally substituted lower alkyl, or a group -D-E, in which D
is a covalent bond or alkylene, and E is optionally substituted phenyl,
optionally substituted cycloalkyl, optionally substituted alkenyl, or
optionally substituted alkynyl, andR3 is hydrogen.

17. The method of claim 16, wherein:X is optionally substituted phenylene;
andY is a covalent bond or lower alkylene in which one carbon atom can be
optionally replaced by --O--, --S--, or --NH--.

26. The method of claim 25, wherein R1 is lower alkyl optionally
substituted by cycloalkyl, R2 is hydrogen, and Y is --CH2-- or
--CH(CH3).

27. The method of claim 26, wherein R1 is n-propyl, X is
1,4-pyrazolene, Y is --CH2--, and Z is 3-trifluoromethylphenyl,
namely 1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]-methyl}pyrazol-4-yl)-1,-
3,7-trihydropurine-2,6-dione.

28. The method of claim 26, wherein R1 is n-propyl, X is
1,4-pyrazolene, Y is --CH2--, and Z is phenyl, namely
1-propyl-8-[1-benzylpyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione.

29. The method of claim 26, wherein R1 is n-butyl, X is
1,4-pyrazolene, Y is --CH2--, and Z is 3-fluorophenyl, namely
1-butyl-8-(1-{[3-fluorophenyl]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2-
,6-dione.

30. The method of claim 26 wherein R1 is n-propyl, X is
1,4-pyrazolene, Y is --CH(CH3)--, and Z is phenyl, namely
1-propyl-8-[1-(phenylethyl)pyrazol-4-yl]-1,3,7-trihydropurine-2,6-dione.

31. The method of claim 25, wherein R1 and R2 are independently
methyl, ethyl, n-propyl, or cyclopropylmethyl, and Y is methylene or
ethylene which may be optionally substituted by hydroxy, alkoxy,
optionally substituted amino, or --COR, which R is hydroxy, alkoxy or
amino.

32. The method of claim 31, wherein R1 and R2 are n-propyl, Y is
--CH2--, and Z is 3-(1,2,3,4-tetrazol-5-yl)phenyl, namely
1,3-dipropyl-8-{1-[(3-(1H-1,2,3,4-tetraazol-5-yl)phenyl)methyl]pyrazol-4--
yl}-1,3,7-trihydropurine-2,6-dione.

33. The method of claim 31, wherein R1 is n-propyl, R2 is ethyl,
Y is --CH2--, and Z is 3-trifluoromethylphenyl, namely
3-ethyl-1-propyl-8-{1-[(3-trifluoromethylphenyl)methyl]pyrazol-4-yl}-1,3,-
7-trihydropurine-2,6-dione.

34. The method of claim 31, wherein R1 and R2 are n-propyl, Y is
--CH(CH3)--, and Z is 3-trifluoromethylphenyl, namely
1,3-dipropyl-8-(1-{[3-(trifluoromethyl)-phenyl]ethyl}pyrazol-4-yl)-1,3,7--
trihydropurine-2,6-dione.

35. The method of claim 31, wherein R1 and R2 are n-propyl, Y is
--CH2--, and Z is 4-carboxyphenyl, namely
1,3-dipropyl-8-{1-[(4-carboxyphenyl)methyl]pyrazol-4-yl}-1,3,7-trihydropu-
rine-2,6-dione.

36. The method of claim 31, wherein R1 and R2 are n-propyl, Y is
--CH2--, and Z is 3-carboxyphenyl, namely
3-{[4-(2,6-dioxo-1,3-dipropyl-1,3,7-trihydropurin-8-yl)pyrazolyl]methyl}b-
enzoic acid.

37. The method of claim 31, wherein R1 and R2 are n-propyl, Y is
--CH(CO2H)--, and Z is phenyl, namely
2-[4-(2,6-dioxo-1,3-dipropyl(1,3,7-trihydropurin-8-yl))pyrazolyl]-2-pheny-
lacetic acid.

60. The method of claim 54, wherein R1 is cyclopropylmethyl, R2
is ethyl, Y is --CH2--, and Z is 6-(trifluoromethyl)-pyrid-3-yl,
namely 1-(cyclopropylmethyl)-3-ethyl-8-(1-{[6-(trifluoromethyl)(3-pyridyl-
)]methyl}pyrazol-4-yl)-1,3,7-trihydropurine-2,6-dione.

61. The method of claim 54, wherein R1 is 2-methylpropyl, R2 is
ethyl, Y is --CH2--, and Z is 6-(trifluoromethyl)-pyrid-3-yl, namely
3-ethyl-1-(2-methylpropyl)-8-(1-{[6-(trifluoromethyl)(3-pyridyl)]methyl}p-
yrazol-4-yl)-1,3,7-trihydropurine-2,6-dione.

Description:

FIELD OF THE INVENTION

[0001]The present invention relates to methods of preventing airway
remodeling and/or pulmonary inflammation using A2B adenosine
receptor antagonists. This invention finds utility in the treatment and
prevention of asthma, COPD, pulmonary fibrosis, emphysema, and other
pulmonary diseases. The invention also relates to pharmaceutical
compositions for use in the method.

BACKGROUND

[0002]According to the Asthma and Allergy Foundation of America and the
National Pharmaceutical Council, an estimated 17 million Americans
currently suffer from asthma. It is the most common chronic childhood
disease, affecting more than one child in 20, nearly 5 million children
in all, and it is the only chronic disease, besides AIDS and
tuberculosis, with an increasing death rate. Each year over 5,000
Americans die from asthma.

[0003]The annual cost of asthma in 1998 was estimated to be $11.3 billion.
Direct costs accounted for $7.5 billion and indirect costs were $3.8
billion. Hospitalizations accounted for the single largest portion of the
cost and amount to nearly a half million hospitalizations, 1.6 million
emergency room visits, and over 10 million office visits. Clearly there
is a great need for new methods of treating the condition.

[0004]As discussed by Elias et al. (1999), J. Clin. Inv.,
104(8):1001-1006, the effects of airway remodeling in the development of
asthma were previously unknown as the condition was thought to be an
entirely reversible disorder. More recent investigations have revealed,
however, that significant airway remodeling occurs during asthma and that
the degree of this remodeling is usually proportional to symptom
severity. Remodeling typically takes the form of airway wall thickening,
the development of subepithelial fibrosis, increased myocyte muscle mass,
myofibroblast hyperplasia, and mucus metaplasia. Airway remodeling is
also a common factor in the progression of chronic obstructive pulmonary
disorder (COPD), and pulmonary fibrosis. Pulmonary inflammation is also a
common component in the development of airway remodeling and may be
typified by bronchiolitis, alveolitis, and/or vasculitis.

[0005]The correlation between airway remodeling and asthma presents a new
avenue of asthma research. Recently the ability of cortical steroids and
leukotrine receptor antagonists to prevent or treat airway remodeling has
been reported (see Hoshino (2004) Clin Rev Allergy Immunol. 27(1):59-64).
Given the potential negative side effects of long term treatment with
cortical steroids and the uncertainties regarding the efficacy of
leukotrine receptor antagonists, the exists a strong need for other
methods of inhibiting airway remodeling.

[0006]Adenosine is known to play a role in asthma and COPD (See, Spicuzza
et al. (2003) TiPS 24(8):409-4130; Mann et al, (1986) J Appl Physiol
61:1667-1676; and Feoktistov et al, (1998) Trends Pharmacol Sci
19:148-153.) The clinical evidence supporting the involvement of
adenosine includes: [0007]1) Plasma concentrations of adenosine are
increased by allergen challenge in asthmatic patients and adenosine
levels in the bronchoalveolar lavage fluid are elevated in asthmatic and
COPD patients (Driver et al, (1993). Am Rev Respir Dis 148:91-97)
[0008]2) Adenosine (given as AMP) induces bronchoconstriction in
asthmatics but not in normal subjects (Cushley et al, (1983) Br J Clin
Pharmacol 15:161-165), and it increases the concentrations of mediators
released from mast cells, such as histamine, tryptase, LTC4 and PDG2
(Crimi et al, (1997) Allergy 52:48-54). The adenosine-induced
bronchoconstriction is attenuated by drugs that either inhibit mast cell
activation or serve as antagonists to the mediators released from the
mast cells. Thus, the potential mechanism of adenosine-induced
bronchoconstriction is likely due to its effect on mast cell activation
(Polosa et al, (2002) Thorax 57:649-654 and Polosa (2002) Eur Respir J
20:488-496.). [0009]3) Adenosine has also been shown to induce
eosinophilia and inflammation. The overall effects and potential clinical
utilities of AMP-challenge are summarized in a recent review article by
Spicuzza and Polosa, (2003) Curr Opin Allergy Clin Immunol 3:65-69.

[0010]Adenosine is a naturally occurring nucleoside, which exerts its
biological effects by interacting with a family of adenosine receptors
known as A1, A2A, A2B, and A3, all of which modulate
important physiological processes. Of the various receptors, A2B
adenosine receptors are believed to be most significantly involved in
asthma via their connection to mast cell activation, vasodilation, and
regulation of cell growth (See Adenosine A2B Receptors as
Therapeutic Targets, Drug Dev Res 45:198; Feoktistov et al., Trends
Pharmacol Sci 19:148-153). Specifically, adenosine A2B receptor
antagonists have been shown to affect the activation of mast cell and
have thus been implicated in the inhibition of the acute airway
hyperresponsiveness. Surprisingly, it has now been found that A2B
adenosine receptor antagonists are also useful in the prevention of
airway remodeling and pulmonary inflammation.

[0011]Accordingly, it is desired to provide a method of preventing airway
remodeling and/or pulmonary inflammation by administration of compounds
that are potent, fully or partially selective, A2B antagonists,
i.e., compounds that inhibit the A2B adenosine receptor.

SUMMARY OF THE INVENTION

[0012]In one embodiment of the invention, a method is provided for the
treatment and prevention of airway remodeling and/or pulmonary
inflammation by administration of a therapeutically effective amount of
an A2B receptor antagonist to a mammal that is genetically and/or
environmentally predisposed to airway remodeling and pulmonary
inflammation.

[0013]In another embodiment of the invention, a method is provided for the
treatment and prevention of airway remodeling and/or pulmonary
inflammation by administration to a mammal that is genetically and/or
environmentally predisposed to airway remodeling, a therapeutically
effective amount of an A2B receptor antagonist having the structure
of Formula I or Formula II:

##STR00001##

wherein: [0014]R1 and R2 are independently chosen from hydrogen,
optionally substituted alkyl, or a group -D-E, in which D is a covalent
bond or alkylene, and E is optionally substituted alkoxy, optionally
substituted cycloalkyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted heterocyclyl, optionally
substituted alkenyl or optionally substituted alkynyl, with the proviso
that when D is a covalent bond E cannot be alkoxy; [0015]R3 is
hydrogen, optionally substituted alkyl or optionally substituted
cycloalkyl; [0016]X is optionally substituted arylene or optionally
substituted heteroarylene; [0017]Y is a covalent bond or alkylene in
which one carbon atom can be optionally replaced by --O--, --S--, or
--NH--, and is optionally substituted by hydroxy, alkoxy, optionally
substituted amino, or --COR, in which R is hydroxy, alkoxy or amino; and
[0018]Z is optionally substituted monocyclic aryl or optionally
substituted monocyclic heteroaryl; or [0019]Z is hydrogen when X is
optionally substituted heteroarylene and Y is a covalent bond.

[0020]In yet another embodiment of the invention, pharmaceutical
formulations are provided, comprising a therapeutically effective amount
of an A2B receptor antagonist, and at least one pharmaceutically
acceptable carrier. The formulation is preferably for oral
administration.

[0021]One preferred group of compounds of Formula I and H are those in
which R1 and R2 are independently hydrogen, optionally
substituted lower alkyl, or a group -D-E, in which D is a covalent bond
or alkylene, and E is optionally substituted phenyl, optionally
substituted cycloalkyl, optionally substituted alkenyl, or optionally
substituted alkynyl, particularly those in which R3 is hydrogen.

[0022]Within this group, a first preferred class of compounds include
those in which R1 and R2 are independently lower alkyl
optionally substituted by cycloalkyl, preferably n-propyl, and X is
optionally substituted phenylene. Within this class, a preferred subclass
of compounds are those in which Y is alkylene, including alkylene in
which a carbon atom is replaced by oxygen, preferably --O--CH2--,
more especially where the oxygen is the point of attachment to phenylene.
Within this subclass, it is preferred that Z is optionally substituted
oxadiazole, particularly optionally substituted [1,2,4]-oxadiazol-3-yl,
especially [1,2,4]-oxadiazol-3-yl substituted by optionally substituted
phenyl or optionally substituted pyridyl.

[0023]A second preferred class of compounds include those in which X is
optionally substituted 1,4-pyrazolene. Within this class, a preferred
subclass of compounds are those in which Y is a covalent bond or
alkylene, especially lower alkylene, and Z is hydrogen, optionally
substituted phenyl, optionally substituted pyridyl, or optionally
substituted oxadiazole. Within this subclass, one preferred embodiment
includes compounds in which R1 is lower alkyl optionally substituted
by cycloalkyl, and R2 is hydrogen. A more preferred embodiment
includes those compounds in which Y is --(CH2)-- or --CH(CH3)--
and Z is optionally substituted phenyl, or Y is --(CH2)-- or
--CH(CH3)-- and Z is optionally substituted oxadiazole, particularly
3,5-[1,2,4]-oxadiazole, or Y is --(CH2)-- or --CH(CH3)-- and Z
is optionally substituted pyridyl. Within this subclass, also preferred
are those compounds in which R1 and R2 are independently lower
alkyl optionally substituted by cycloalkyl, especially n-propyl. More
preferred are those compounds in which Y is a covalent bond,
--(CH2)-- or --CH(CH3)-- and Z is hydrogen, optionally
substituted phenyl, or optionally substituted pyridyl, particularly where
Y is a covalent bond and Z is hydrogen.

[0063]FIG. 2 shows the effects of an adenosine A2B receptor
antagonist (A2B-A) on airway cellularity as described in Example 21.
(A) Mice were lavalged with PBS and total cell counts determined. (B and
C) Cells were cytospun onto microscope slides, stained with DiffQuick and
cellular differentials were determined by counting at least 200 cells per
sample. Values are presented as mean total cells×104±SEM.
*, significant at p<0.05 compared to ADA+ animals using the
students T test, n=8; #, significant at p<0.05 compared to
vehicle-treated ADA.sup.-/- mice using the students T test, n=6-8.

[0068]FIG. 7 shows the expression of fibrosis associated genes. RNA was
extracted from whole lungs for analysis using quantitative RT-PCR for
various fibrosis associated transcripts. Results demonstrate that lungs
from an adenosine A2B receptor antagonist treated ADA.sup.-/- mice
exhibit lower levels of transcripts for TGF-β1 (A), osteopontin
(OPN) (B), and plasminogen activator inhibitor-1 (Pai-1) (C), as compared
to that seen in the lungs of vehicle treated ADA.sup.-/- mice. *,
significant at p<0.05 compared to ADA+ mice using the students T
test, #, significant at p<0.05 compared to vehicle-treated ADA.sup.-/-
mice using the students T test, n=4 for ADA+ mice, n=8 for ADA mice.

[0070]FIG. 9 presents alveolar destruction in ADA-/- mice treated with an
adenosine A2B receptor antagonist as described in Example 21. Lungs
were collected and processed for histological analysis using H&E
staining. (A) Lung from ADA+ vehicle treated mouse, (B) Lung from ADA-/-
vehicle treated mouse, and (C) Lung from ADA-/- mouse treated with an
adenosine A2B receptor antagonist. Photographs were all taken at the
same magnification (10×) and represent findings from 6 different
animals for each treatment group. (D) Alveolar airway sizes were
calculated using ImagePro and data is presented as mean cord
length±SEM, n=6. In (D), * denotes that the results are significantly
different form ADA+ mice and ** denotes that results are significantly
different from ADA-/- vehicle treated mice.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Parameters

[0071]As used in the present specification, the following words and
phrases are generally intended to have the meanings as set forth below,
except to the extent that the context in which they are used indicates
otherwise.

[0073]The term "substituted alkyl" refers to: [0074]1) an alkyl group as
defined above, having 1, 2, 3, 4 or 5 substituents, preferably 1 to 3
substituents, selected from the group consisting of alkenyl, alkynyl,
alkoxy, cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy, amino,
aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen, hydroxy, keto,
thiocarbonyl, carboxy, carboxyalkyl, arylthio, heteroarylthio,
heterocyclylthio, thiol, alkylthio, aryl, aryloxy, heteroaryl,
aminosulfonyl, aminocarbonylamino, heteroaryloxy, heterocyclyl,
heterocyclooxy, hydroxyamino, alkoxyamino, nitro, --SO-alkyl, --SO-aryl,
--SO-heteroaryl, --SO2-alkyl, SO2-aryl and
--SO2-heteroaryl. Unless otherwise constrained by the definition,
all substituents may optionally be further substituted by 1, 2, or 3
substituents chosen from alkyl, carboxy, carboxyalkyl, aminocarbonyl,
hydroxy, alkoxy, halogen, CF3, amino, substituted amino, cyano, and
--S(O)nR, where R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2;
or [0075]2) an alkyl group as defined above that is interrupted by 1-10
atoms independently chosen from oxygen, sulfur and NRa--, where
Ra is chosen from hydrogen, alkyl, cycloalkyl, alkenyl,
cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclyl. All
substituents may be optionally further substituted by alkyl, alkoxy,
halogen, CF3, amino, substituted amino, cyano, or --S(O)nR, in
which R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2; or [0076]3) an
alkyl group as defined above that has both 1, 2, 3, 4 or 5 substituents
as defined above and is also interrupted by 1-10 atoms as defined above.

[0077]The term "lower alkyl" refers to a monoradical branched or
unbranched saturated hydrocarbon chain having 1, 2, 3, 4, 5, or 6 carbon
atoms. This term is exemplified by groups such as methyl, ethyl,
n-propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, n-hexyl, and the like.

[0078]The term "substituted lower alkyl" refers to lower alkyl as defined
above having 1 to 5 substituents, preferably 1, 2, or 3 substituents, as
defined for substituted alkyl, or a lower alkyl group as defined above
that is interrupted by 1, 2, 3, 4, or 5 atoms as defined for substituted
alkyl, or a lower alkyl group as defined above that has both 1, 2, 3, 4
or 5 substituents as defined above and is also interrupted by 1, 2, 3, 4,
or 5 atoms as defined above.

[0086]The term "aralkyl" refers to an aryl group covalently linked to an
alkylene group, where aryl and alkylene are defined herein. "Optionally
substituted aralkyl" refers to an optionally substituted aryl group
covalently linked to an optionally substituted alkylene group. Such
aralkyl groups are exemplified by benzyl, phenylethyl,
3-(4-methoxyphenyl)propyl, and the like.

[0087]The term "alkoxy" refers to the group R--O--, where R is optionally
substituted alkyl or optionally substituted cycloalkyl, or R is a group
--Y--Z, in which Y is optionally substituted alkylene and Z is optionally
substituted alkenyl, optionally substituted alkynyl; or optionally
substituted cycloalkenyl, where alkyl, alkenyl, alkynyl, cycloalkyl and
cycloalkenyl are as defined herein. Preferred alkoxy groups are
optionally substituted alkyl-O-- and include, by way of example, methoxy,
ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,
n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, trifluoromethoxy, and the like.

[0088]The term "alkylthio" refers to the group R--S--, where R is as
defined for alkoxy.

[0089]The term "alkenyl" refers to a monoradical of a branched or
unbranched unsaturated hydrocarbon group preferably having from 2 to 20
carbon atoms, more preferably 2 to 10 carbon atoms and even more
preferably 2 to 6 carbon atoms and having 1-6, preferably 1, double bond
(vinyl). Preferred alkenyl groups include ethenyl or vinyl
(--CH═CH2), 1-propylene or allyl (--CH2CH═CH2),
isopropylene (--C(CH3)═CH2), bicyclo[2.2.1]heptene, and the
like. In the event that alkenyl is attached to nitrogen, the double bond
cannot be alpha to the nitrogen.

[0090]The term "lower alkenyl" refers to alkenyl as defined above having
from 2 to 6 carbon atoms.

[0092]The term "alkynyl" refers to a monoradical of an unsaturated
hydrocarbon, preferably having from 2 to 20 carbon atoms, more preferably
2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and
having at least 1 and preferably from 1-6 sites of acetylene (triple
bond) unsaturation. Preferred alkynyl groups include ethynyl, propargyl
(or prop-1-yn-3-yl, --CH2C≡CH), and the like. In the event
that alkynyl is attached to nitrogen, the triple bond cannot be alpha to
the nitrogen.

[0094]The term "aminocarbonyl" refers to the group --C(O)NRR where each R
is independently hydrogen, alkyl, aryl, heteroaryl, heterocyclyl or where
both R groups are joined to form a heterocyclic group (e.g., morpholino).
Unless otherwise constrained by the definition, all substituents may
optionally be further substituted by 1-3 substituents chosen from alkyl,
carboxy, carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF3,
amino, substituted amino, cyano, and S(O)nR, where R is alkyl, aryl,
or heteroaryl and n is 0, 1 or 2.

[0095]The term "acylamino" refers to the group --NRC(O)R where each R is
independently hydrogen, alkyl, aryl, heteroaryl, or heterocyclyl. Unless
otherwise constrained by the definition, all substituents may optionally
be further substituted by 1-3 substituents chosen from alkyl, carboxy,
carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF3, amino,
substituted amino, cyano, and --S(O)nR, where R is alkyl, aryl, or
heteroaryl and n is 0, 1 or 2.

[0096]The term "acyloxy" refers to the groups --O(O)C-alkyl,
O(O)C-cycloalkyl, O(O)C-aryl, --O(O)C-heteroaryl, and
--O(O)C-heterocyclyl. Unless otherwise constrained by the definition, all
substituents may be optionally further substituted by alkyl, carboxy,
carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF3, amino,
substituted amino, cyano, or S(O)nR, where R is alkyl, aryl, or
heteroaryl and n is 0, 1 or 2.

[0097]The term "aryl" refers to an aromatic carbocyclic group of 6 to 20
carbon atoms having a single ring (e.g., phenyl) or multiple rings (e.g.,
biphenyl), or multiple condensed (fused) rings (e.g., naphthyl or
anthryl). Preferred aryls include phenyl, naphthyl and the like.

[0098]The term "arylene" refers to a diradical of an aryl group as defined
above. This term is exemplified by groups such as 1,4-phenylene,
1,3-phenylene, 1,2-phenylene, 1,4'-biphenylene, and the like.

[0100]The term "aryloxy" refers to the group aryl-O-- wherein the aryl
group is as defined above, and includes optionally substituted aryl
groups as also defined above. The term "arylthio" refers to the group
R--S--, where R is as defined for aryl.

[0101]The term "amino" refers to the group --NH2.

[0102]The term "substituted amino" refers to the group --NRR where each R
is independently selected from the group consisting of hydrogen, alkyl,
cycloalkyl, carboxyalkyl (for example, benzyloxycarbonyl), aryl,
heteroaryl and heterocyclyl provided that both R groups are not hydrogen,
or a group --Y--Z, in which Y is optionally substituted alkylene and Z is
alkenyl, cycloalkenyl, or alkynyl, Unless otherwise constrained by the
definition, all substituents may optionally be further substituted by 1-3
substituents chosen from alkyl, carboxy, carboxyalkyl, aminocarbonyl,
hydroxy, alkoxy, halogen, CF3, amino, substituted amino, cyano, and
S(O)nR, where R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2.

[0103]The term "carboxyalkyl" refers to the groups --C(O)O-alkyl or
--C(O)O-cycloalkyl, where alkyl and cycloalkyl, are as defined herein,
and may be optionally further substituted by alkyl, alkenyl, alkynyl,
alkoxy, halogen, CF3, amino, substituted amino, cyano, or
S(O)nR, in which R is alkyl, aryl, or heteroaryl and n is 0, 1 or 2.

[0104]The term "cycloalkyl" refers to carbocyclic groups of from 3 to 20
carbon atoms having a single cyclic ring or multiple condensed rings.
Such cycloalkyl groups include, by way of example, single ring structures
such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like,
or multiple ring structures such as adamantanyl, bicyclo[2.2.1]heptane,
1,3,3-trimethylbicyclo[2.2.1]kept-2-yl,
(2,3,3-trimethylbicyclo[2.2.1]hept-2-yl), or carbocyclic groups to which
is fused an aryl group, for example indane, and the like.

[0109]The term "heteroarylene" refers to a diradical of a heteroaryl group
as defined above. This teen is exemplified by groups such as
2,5-imidazolene, 3,5-[1,2,4]oxadiazolene, 2,4-oxazolene, 1,4-pyrazolene,
and the like. For example, 1,4-pyrazolene is:

[0111]The term "heteroaralkyl" refers to a heteroaryl group covalently
linked to an alkylene group, where heteroaryl and alkylene are defined
herein. "Optionally substituted heteroaralkyl" refers to an optionally
substituted heteroaryl group covalently linked to an optionally
substituted alkylene group. Such heteroaralkyl groups are exemplified by
3-pyridylmethyl, quinolin-8-ylethyl, 4-methoxythiazol-2-ylpropyl, and the
like.

[0112]The term "heteroaryloxy" refers to the group heteroaryl-O--.

[0113]The term "heterocyclyl" refers to a monoradical saturated or
partially unsaturated group having a single ring or multiple condensed
rings, having from 1 to 40 carbon atoms and from 1 to 10 hetero atoms,
preferably 1, 2, 3 or 4 heteroatoms, selected from nitrogen, sulfur,
phosphorus, and/or oxygen within the ring. Heterocyclic groups can have a
single ring or multiple condensed rings, and include tetrahydrofuranyl,
morpholino, piperidinyl, piperazino, dihydropyridino, and the like.

[0116]The term "substituted alkylthio" refers to the group S-substituted
alkyl.

[0117]The term "heteroarylthio" refers to the group S-heteroaryl wherein
the heteroaryl group is as defined above including optionally substituted
heteroaryl groups as also defined above.

[0118]The term "sulfoxide" refers to a group --S(O)R, in which R is alkyl,
aryl, or heteroaryl. "Substituted sulfoxide" refers to a group --S(O), in
which R is substituted alkyl, substituted aryl, or substituted
heteroaryl, as defined herein.

[0119]The term "sulfone" refers to a group --S(O)2R, in which R is
alkyl, aryl, or heteroaryl. "Substituted sulfone" refers to a group
--S(O)2R, in which R1 is substituted alkyl, substituted aryl,
or substituted heteroaryl, as defined herein.

[0120]The term "keto" refers to a group --C(O)--.

[0121]The term "thiocarbonyl" refers to a group --C(S)--.

[0122]The term "carboxy" refers to a group --C(O)--OH.

[0123]"Optional" or "optionally" means that the subsequently described
event or circumstance may or may not occur, and that the description
includes instances where said event or circumstance occurs and instances
in which it does not.

[0124]The term "compound of Formula I and Formula II" is intended to
encompass the compounds of the invention as disclosed, and the
pharmaceutically acceptable salts, pharmaceutically acceptable esters,
prodrugs, hydrates and polymorphs of such compounds. Additionally, the
compounds of the invention may possess one or more asymmetric centers,
and can be produced as a racemic mixture or as individual enantiomers or
diastereoisomers. The number of stereoisomers present in any given
compound of Formula I depends upon the number of asymmetric centers
present (there are 2n stereoisomers possible where n is the number
of asymmetric centers). The of an intermediate at some appropriate stage
of the synthesis, or by resolution of the compound of Formula I by
conventional means. The individual stereoisomers (including individual
enantiomers and diastereoisomers) as well as racemic and non-racemic
mixtures of stereoisomers are encompassed within the scope of the present
invention, all of which are intended to be depicted by the structures of
this specification unless otherwise specifically indicated.

[0125]"Isomers" are different compounds that have the same molecular
formula.

[0126]"Stereoisomers" are isomers that differ only in the way the atoms
are arranged in space.

[0127]"Enantiomers" are a pair of stereoisomers that are
non-superimposable mirror images of each other. A 1:1 mixture of a pair
of enantiomers is a "racemic" mixture. The term "(±)" is used to
designate a racemic mixture where appropriate.

[0128]"Diastereoisomers" are stereoisomers that have at least two
asymmetric atoms, but which are not mirror-images of each other.

[0129]The absolute stereochemistry is specified according to the
Cahn-Ingold-Prelog R--S system. When the compound is a pure enantiomer
the stereochemistry at each chiral carbon may be specified by either R or
S. Resolved compounds whose absolute configuration is unknown are
designated (+) or (-) depending on the direction (dextro- or laevorotary)
which they rotate the plane of polarized light at the wavelength of the
sodium D line.

[0130]"Topical administration" shall be defined as the delivery of the
therapeutic agent to the surface of the wound and adjacent epithelium.

[0131]"Parenteral administration" is the systemic delivery of the
therapeutic agent via injection to the patient.

[0132]The term "therapeutically effective amount" refers to that amount of
a compound of Formula I that is sufficient to effect treatment, as
defined below, when administered to a mammal in need of such treatment.
The therapeutically effective amount will vary depending upon the
specific activity of the therapeutic agent being used, and the age,
physical condition, existence of other disease states, and nutritional
status of the patient. Additionally, other medication the patient may be
receiving will effect the determination of the therapeutically effective
amount of the therapeutic agent to administer.

[0133]The term "treatment" or "treating" means any treatment of a disease
in a mammal, including: [0134](i) preventing the disease, that is,
causing the clinical symptoms of the disease not to develop; [0135](ii)
inhibiting the disease, that is, arresting the development of clinical
symptoms; and/or [0136](iii) relieving the disease, that is, causing the
regression of clinical symptoms.

[0137]The language "genetically and/or environmentally predisposed to
airway remodeling and/or pulmonary inflammation" refers to mammals that
are susceptible to Examples of such environmental conditions include, but
are not limited to, exposure to cigarette smoke and other pollutants,
exposure to sprays or chemical agents at work, home, or with hobbies,
exposure to common allergens such as dust, grasses, molds, weeds, trees,
and animal dander, and exposure to irritants such as asbestos, silica and
metal dusts. Examples of genetic predisposition can be evidenced by
family history or genetic analysis for suspected mutations in the ADAM33
gene, TLR4 polymorphisms, IL-3 polymorphisms, CD14 C-159T
polymorphisms, and the like. Bioinformatic methods of screening for a
genetic predisposition have been presented by Tomita et al. (2004) BMC
Bioinformatics. 5(1):120. Other conditions and disease states that are
known to cause airway remodeling and/or pulmonary inflammation include,
but are not limited to, lupus, scleroderma, tuberculosis, and rheumatoid
arthritis.

[0138]In many cases, the compounds of this invention are capable of
forming acid and/or base salts by virtue of the presence of amino and/or
carboxyl groups or groups similar thereto. The term "pharmaceutically
acceptable salt" refers to salts that retain the biological effectiveness
and properties of the compounds of Formula I, and which are not
biologically or otherwise undesirable. Pharmaceutically acceptable base
addition salts can be prepared from inorganic and organic bases. Salts
derived from inorganic bases, include by way of example only, sodium,
potassium, lithium, ammonium, calcium and magnesium salts. Salts derived
from organic bases include, but are not limited to, salts of primary,
secondary and tertiary amines, such as alkyl amines, dialkyl amines,
trialkyl amines, substituted alkyl amines, di(substituted alkyl) amines,
tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines,
trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl)
amines, tri(substituted alkenyl) amines, cycloalkyl amines,
di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkyl
amines, disubstituted cycloalkyl amine, trisubstituted cycloalkyl amines,
cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl) amines,
substituted cycloalkenyl amines, disubstituted cycloalkenyl amine,
trisubstituted cycloalkenyl amines, aryl amines, diaryl amines, triaryl
amines, heteroaryl amines, diheteroaryl amines, triheteroaryl amines,
heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed
di- and tri-amines where at least two of the substituents on the amine
are different and are selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted
cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl,
heterocyclic, and the like. Also included are amines where the two or
three substituents, together with the amino nitrogen, form a heterocyclic
or heteroaryl group.

[0141]As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents and the like.
The use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media or
agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.

Nomenclature

[0142]The naming and numbering of the compounds of the invention is
illustrated with a representative compound of Formula I in which R1
is n-propyl, R2 is n-propyl, R3 is hydrogen, X is phenylene, Y
is --O--(CH2), and Z is 5-(2-methoxyphenyl)-[1,2,4]-oxadiazol-3-yl,

##STR00003##

which is named:
[0143]8-{4-[5-(2-methoxyphenyl)-[1,2,4]-oxadiazol-3-ylmethoxy]-phenyl}-1,-
3-dipropyl-1,3,7-trihydropurine-2,6-dione.

The Method of the Invention

[0144]The present invention relates to methods of preventing airway
remodeling and/or pulmonary inflammation by administration of a
therapeutically effective amount of a A2B adenosine receptor
antagonist to a mammal having a condition that produces or is caused by
such airway remodeling. As airway remodeling and pulmonary inflammation
are significant components of asthma, pulmonary fibrosis, and COPD, the
method of the invention will generally involve administration of an
A2B adenosine receptor antagonist to a patient suffering from either
asthma, pulmonary fibrosis, and/or COPD.

[0145]The A2B adenosine receptor antagonist is administered
systemically as an oral formulation but may also be administered directly
to the pulmonary tissue via an inhaler. This administration can be as a
single dose or as repeated doses given at multiple designated intervals.
It will readily be appreciated by those skilled in the art that the
preferred dosage regimen will vary with the patient and severity of the
condition being treated.

Pharmaceutical Compositions

[0146]When selected as the adenosine A2B receptor antagonist, the
compounds of Formula I are usually administered in the form of
pharmaceutical compositions. This invention therefore provides
pharmaceutical compositions that contain, as the active ingredient, one
or more of the compounds of Formula I, or a pharmaceutically acceptable
salt or ester thereof, and one or more pharmaceutically acceptable
excipients, carriers, including inert solid diluents and fillers,
diluents, including sterile aqueous solution and various organic
solvents, solubilizers and adjuvants. The compounds of Formula I may be
administered alone or in combination with other therapeutic agents. Such
compositions are prepared in a manner well known in the pharmaceutical
art (see, e.g., Remington's Pharmaceutical Sciences, Mace Publishing Co.,
Philadelphia, Pa. 17th Ed. (1985) and "Modern Pharmaceutics", Marcel
Dekker, Inc. 3rd Ed. (G. S. Banker & C. T. Rhodes, Eds.).

The A2B Adenosine Receptor Antagonists

[0147]Any A2B adenosine receptor antagonist may be used in the method
of the invention. Numerous compounds that antagonize the A2B
receptor are known in the art, as are methods for determining if a
specific compound has such activity. For example, a review article by
Feoktistov and Baggioni, ((1997) Pharmacological Reviews 49:381-402)
reports the binding affinity of eight adenosine receptor agonists and
eight antagonists for all four subtypes of adenosine receptors.
References cited therein provide detailed descriptions of the procedures
used. (Robeva et al, (1996) J. Drug Dev, Res 39:243-252; Jacobson et al
(1996) Drug Dev. Res, 39:289-300; Feoktistov and Baggioni (1993)
Molecular Pharmacology 43:909-914). Effective methods for determining the
binding affinity of a compound for a receptor use a radiolabelled agonist
or antagonist and correlation of the binding of that compound to a
membrane fraction known to contain that receptor; for example, to
determine whether a compound is an A2B antagonist, the membrane
fraction would contain the A2B adenosine receptor. Another
particularly effective procedure for determining whether a compound is an
A2B antagonist is reported in U.S. Pat. No. 5,854,081.

[0148]Compounds selective for the A2B receptor subtype are therefore
preferred for the present methods. An example, but not a limitation, of
such a compound is 3-n-propylxanthine (enprofylline). Suitable compounds
are also disclosed in U.S. Pat. No. 6,545,002. Compounds that antagonize
other receptors in addition to the A2B receptor are also suitable
for use in the present invention. One example of such a compound is
1,3-dipropyl-8-(p-acrylic)phenylxanthine.

[0149]One particularly preferred class of A2B adenosine receptor
antagonists are those disclosed in copending and commonly assigned U.S.
Pat. No. 6,825,349 and in copending and commonly assigned U.S. patent
application Ser. No. 10/719,102, which published as U.S. Patent
Application Publication No. 20040176399. The compounds disclosed in that
application have the structure of Formula I and Formula II as presented
in the Summary of the Invention above and can be synthesized as described
in the references or as detailed below.

Synthetic Reaction Parameters

[0150]The terms "solvent", "inert organic solvent" or "inert solvent" mean
a solvent inert under the conditions of the reaction being described in
conjunction therewith [including, for example, benzene, toluene,
acetonitrile, tetrahydrofuran ("THF"), dimethylformamide ("DMF"),
chloroform, methylene chloride (or dichloromethane), diethyl ether,
methanol, pyridine and the like]. Unless specified to the contrary, the
solvents used in the reactions of the present invention are inert organic
solvents, and the reactions are carried out under an inert gas,
preferably nitrogen.

[0151]The term "q.s." means adding a quantity sufficient to achieve a
stated function, e.g., to bring a solution to the desired volume (i.e.,
100%).

Synthesis of the Compounds of Formula I and II

[0152]One preferred method of preparing compounds of Formula I or II where
R3 is hydrogen is shown in Reaction Scheme I.

##STR00004##

Step 1--Preparation of Formula (2)

[0153]The compound of formula (2) is made from the compound of formula (1)
by a reduction step. Conventional reducing techniques may be used, for
example using sodium dithionite in aqueous ammonia solution; preferably,
reduction is carried out with hydrogen and a metal catalyst. The reaction
is carried out at in an inert solvent, for example methanol, in the
presence of a catalyst, for example 10% palladium on carbon catalyst,
under an atmosphere of hydrogen, preferably under pressure, for example
at about 30 psi, for about 2 hours. When the reaction is substantially
complete, the product of formula (2) is isolated by conventional means to
provide a compound of formula (2).

Step 2--Preparation of Formula (3)

[0154]The compound of formula (2) is then reacted with a carboxylic acid
of the formula Z--Y--X--CO2H in the presence of a carbodiimide, for
example 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The
reaction is conducted in a protic solvent, for example methanol, ethanol,
propanol, and the like, preferably methanol, at a temperature of about
20-30° C., preferably about room temperature, for about 12-48
hours, preferably about 16 hours. When the reaction is substantially
complete, the product of formula (3) is isolated conventionally, for
example by removal of the solvent under reduced pressure, and washing the
product. Alternatively, the next step can be carried out without any
further purification.

Alternative Preparation of a Compound of Formula (3)

[0155]Alternatively, the carboxylic acid of the formula Z--Y--X--CO2H
is first converted to an acid halide of the formula Z--Y--X--C(O)L, where
L is chloro or bromo, by reacting with a halogenating agent, for example
thionyl chloride or thionyl bromide, preferably thiony chloride.
Alternatively, oxalyl chloride, phosphorus pentachloride or phosphorus
oxychloride may be used. The reaction is preferably conducted in the
absence of a solvent, using excess halogenating agent, for example at a
temperature of about 60-80° C., preferably about 70° C.,
for about 1-8 hours, preferably about 4 hours. When the reaction is
substantially complete, the product of formula Z--Y--X--C(O)L is isolated
conventionally, for example by removal of the excess halogenating agent
under reduced pressure.

[0156]The product is then reacted with a compound of formula (2) in an
inert solvent, for example acetonitrile, in the presence of a tertiary
base, for example triethylamine. The reaction is conducted at an initial
temperature of about 0C, and then allowed to warm to 20-30° C.,
preferably about room temperature, for about 12-48 hours, preferably
about 16 hours. When the reaction is substantially complete, the product
of formula (3) is isolated conventionally, for example by diluting the
reaction mixture with water, filtering off the product, and washing the
product with water followed by ether.

Step 3--Preparation of Formula I

[0157]The compound of formula (3) is then converted into a compound of
Formula I by a cyclization reaction. The reaction is conducted in a
protic solvent, for example methanol, ethanol, propanol, and the like,
preferably methanol, in the presence of a base, for example potassium
hydroxide, sodium hydroxide, sodium methoxide, sodium ethoxide, potassium
t-butoxide, preferably aqueous sodium hydroxide, at a temperature of
about 50-80° C., preferably about 80° C., for about 1-8
hours, preferably about 3 hours. When the reaction is substantially
complete, the product of Formula I is isolated conventionally, for
example by removal of the solvent under reduced pressure, acidifying the
residue with an aqueous acid, filtering off the product, then washing and
drying the product.

[0158]The compound of formula (1) may be prepared by various methods. One
preferred method is shown in Reaction Scheme II.

##STR00005##

Step 1--Preparation of Formula (5)

[0159]The compound of formula (4) is either commercially available or
prepared by means well known in the art. It is reacted with ethyl
cyanoacetate in a protic solvent, for example ethanol, in the presence of
a strong base, for example sodium ethoxide. The reaction is carried out
at about reflux temperature, for about 4 to about 24 hours. When the
reaction is substantially complete, the compound of formula (5) thus
produced is isolated conventionally.

Step 2 and 3--Preparation of Formula (7)

[0160]The compound of formula (5) is reacted with the dimethylacetal of
N,N-dimethylformamide in a polar solvent, for example
N,N-dimethylformamide. The reaction is carried out at about 40°
C., for about 1 hour. When the reaction is substantially complete, the
compound of formula (6) thus produced is reacted with a compound of
formula R1Hal, where Hal is chloro, bromo, or iodo, in the presence
of a base, for example potassium carbonate. The reaction is carried out
at about 80° C., for about 4-24 hour. When the reaction is
substantially complete, the product of formula (7) is isolated
conventionally, for example by evaporation of the solvents under reduced
pressure, and the residue is used in the next reaction with no further
purification.

Step 4--Preparation of Formula (8)

[0161]The compound of formula (7) is reacted with aqueous ammonia in a
polar solvent, for example suspended in methanol. The reaction is carried
out at about room temperature, for about 1-3 days. When the reaction is
substantially complete, the product of formula (8) is isolated
conventionally, for example by chromatography over a silica gel column,
eluting, for example, with a mixture of dichloromethane/methanol.

Step 5--Preparation of Formula (1)

[0162]The compound of formula (8) is then mixed with sodium nitrite in an
aqueous acidic solvent, preferably acetic acid and water, for example 50%
acetic acid/water. The reaction is carried out at a temperature of about
50-90° C., preferably about 70° C., for about 1 hour. When
the reaction is substantially complete, the product of formula (1) is
isolated by conventional means.

[0163]Alternatively, the reaction may be conducted in an aqueous solvent,
for example dimethylformamide and water, and reacted with a strong acid,
for example hydrochloric acid.

[0164]A compound of formula (8) can be prepared from a compound of formula
(10) using a similar method, as shown in Reaction Scheme IIA.

##STR00006##

Step 2 and 3--Preparation of Formula (7)

[0165]The compound of formula (10) is reacted with the dimethylacetal of
N,N-dimethylformamide in a polar solvent, for example
N,N-dimethylfounamide. The reaction is carried out at about 40°
C., for about 1 hour. When the reaction is substantially complete, the
compound of formula (6a) thus produced is reacted with a compound of
formula R2Hal, where Hal is chloro, bromo, or iodo, in the presence
of a base, for example potassium carbonate. The reaction is carried out
at about 80° C., for about 4-24 hour. When the reaction is
substantially complete, the product of formula (7) is isolated
conventionally, for example by evaporation of the solvents under reduced
pressure, and the residue is used in the next reaction with no further
purification.

Step 4--Preparation of Formula (8)

[0166]The compound of formula (7) is reacted with aqueous ammonia in a
polar solvent, for example suspended in methanol. The reaction is carried
out at about room temperature, for about 1-3 days. When the reaction is
substantially complete, the product of formula (8) is isolated
conventionally, for example by chromatography over a silica gel column,
eluting, for example, with a mixture of dichloromethane/methanol.

[0167]The compound of formula (3) may also be prepared by various methods.
One preferred method is shown in Reaction Scheme III.

##STR00007##

Step 1--Preparation of Formula (10)

[0168]The commercially available compound 6-aminouracil is first
silylated, for example by reaction with excess hexamethyldisilazane as a
solvent in the presence of a catalyst, for example ammonium sulfate. The
reaction is carried out at about reflux temperature, for about 1-10
hours. When the reaction is substantially complete, the silylated
compound thus produced is isolated conventionally, and then reacted with
a compound of formula R1Hal, where Hal is chloro, bromo, or iodo,
preferably in the absence of a solvent. The reaction is carried out at
about reflux, for about 4-48 hours, preferably about 12-16 hours. When
the reaction is substantially complete, the product of formula (10) is
isolated by conventional means.

Step 2--Preparation of Formula (11)

[0169]The compound of formula (10) is then dissolved in an aqueous acid,
for example aqueous acetic acid, and reacted with sodium nitrite. The
reaction is carried out at a temperature of about 20-50° C.,
preferably about 30° C., over about 30 minutes. When the reaction
is substantially complete, the product of formula (11) is isolated by
conventional means, for example by filtration.

Step 3--Preparation of Formula (12)

[0170]The compound of formula (11) is then reduced to a diamino
derivative. In general, the compound of formula (11) is dissolved in
aqueous ammonia, and then a reducing agent, for example sodium
hydrosulfite, added. The reaction is conducted at a temperature of about
70° C. When the reaction is substantially complete, the product of
formula (12) is isolated conventionally, for example by filtration of the
cooled reaction mixture.

Step 4--Preparation of Formula (13)

[0171]The compound of formula (12) is then reacted with a carboxylic acid
of the formula Z--Y--X--CO2H in the presence of a carbodiimide, for
example 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The
reaction is conducted at a temperature of about 20-30° C., for
about 12-48 hours. When the reaction is substantially complete, the
product of formula (13) is isolated conventionally, for example by
filtration of the cooled reaction mixture.

[0172]Alternatively, the carboxylic acid of the formula Z--Y--X--CO2H
is converted to an acid halide of the formula Z--Y--X--C(O)L, where L is
chloro or bromo, by reacting with a halogenating agent, for example
thionyl chloride or thionyl bromide; alternatively, phosphorus
pentachloride or phosphorus oxychloride may be used. The reaction is
preferably conducted in the absence of a solvent, using excess
halogenating agent, for example at a temperature of about 60-80°
C., preferably about 70° C., for about 1-8 hours, preferably about
4 hours. When the reaction is substantially complete, the product of
formula Z--Y--X--C(O)L is isolated conventionally, for example by removal
of the excess halogenating agent under reduced pressure.

[0173]The product of the formula Z--Y--X--C(O)L is then reacted with a
compound of formula (12) in an inert solvent, for example acetonitrile,
in the presence of a tertiary base, for example triethylamine. The
reaction is conducted at an initial temperature of about 0C, and then
allowed to warm to 20-30° C., preferably about room temperature,
for about 12-48 hours, preferably about 16 hours. When the reaction is
substantially complete, the product of formula (13) is isolated
conventionally, for example by diluting the reaction mixture with water,
filtering off the product, and washing the product with water followed by
ether.

Step 5--Preparation of Formula (3)

[0174]The compound of formula (13) is reacted with a compound of formula
R2Hal, where Hal is chloro, bromo, or iodo, in the presence of a
base, for example potassium carbonate. The reaction is carried out at
about room temperature, for about 4-24 hour, preferably about 16 hours.
When the reaction is substantially complete, the product of formula (3)
is isolated conventionally, for example by evaporation of the solvents
under reduced pressure, and the residue may be purified conventionally,
or may be used in the next reaction with no further purification.

[0175]Another method of preparing a compound of formula (3) is shown in
Reaction Scheme IV.

##STR00008##

Step 1--Preparation of Formula (14)

[0176]The compound of formula (5) is then mixed with sodium nitrite in an
aqueous acidic solvent, preferably acetic acid and water, for example 50%
acetic acid/water. The reaction is carried out at a temperature of about
50-90° C., preferably about 70° C., for about 1 hour. When
the reaction is substantially complete, the product of formula (14) is
isolated by conventional means.

[0177]Alternatively, the reaction may be conducted in an aqueous solvent,
for example dimethylformamide and water, and reacted with a strong acid,
for example hydrochloric acid.

Step 3--Preparation of Formula (15)

[0178]The compound of formula (14) is then reduced to a diamino
derivative. In general, the compound of formula (14) is dissolved in
aqueous ammonia, and then a reducing agent, for example sodium
hydrosulfite, added. The reaction is conducted at a temperature of about
70° C. When the reaction is substantially complete, the product of
formula (15) is isolated conventionally, for example by filtration of the
cooled reaction mixture.

Step 4--Preparation of Formula (16)

[0179]The compound of formula (15) is then reacted with a carboxylic acid
of the formula Z--Y--X--CO2H in the presence of a carbodiimide, for
example 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The
reaction is conducted at a temperature of about 20-30° C., for
about 12-48 hours, in an inert solvent, for example methanol. When the
reaction is substantially complete, the product of formula (16) is
isolated conventionally, for example by filtration of the cooled reaction
mixture.

[0180]Alternatively, the carboxylic acid of the formula Z--Y--X--CO2H
is converted to an acid halide of the formula Z--Y--X--C(O)L, where L is
chloro or bromo, by reacting with a halogenating agent, for example
thionyl chloride or thionyl bromide; alternatively, phosphorus
pentachloride or phosphorus oxychloride may be used. The reaction is
preferably conducted in the absence of a solvent, using excess
halogenating agent, for example at a temperature of about 60-80°
C., preferably about 70° C., for about 1-8 hours, preferably about
4 hours. When the reaction is substantially complete, the product of
formula Z--Y--X--C(O)L is isolated conventionally, for example by removal
of the excess halogenating agent under reduced pressure.

[0181]The product of the formula Z--Y--X--C(O)L is then reacted with a
compound of formula (15) in an inert solvent, for example acetonitrile,
in the presence of a tertiary base, for example triethylamine. The
reaction is conducted at an initial temperature of about 0C, and then
allowed to warm to 20-30° C., preferably about room temperature,
for about 12-48 hours, preferably about 16 hours. When the reaction is
substantially complete, the product of formula (16) is isolated
conventionally, for example by diluting the reaction mixture with water,
filtering off the product, and washing the product with water followed by
ether.

Step 5--Preparation of Formula (3)

[0182]The compound of formula (16) is reacted with a compound of formula
R1 Hal, where Hal is chloro, bromo, or iodo, in the presence of a
base, for example potassium carbonate. The reaction is carried out at
about 80° C., for about 4-24 hour, preferably about 16 hours. When
the reaction is substantially complete, the product of formula (3) is
isolated conventionally, for example by evaporation of the solvents under
reduced pressure, and the residue may be purified conventionally, or may
be used in the next reaction with no further purification.

[0183]An example of a synthesis of a compound of Z--Y--X--CO2H in
which X is pyrazol-1,4-yl, Y is methylene, and Z is
3-trifluoromethylphenyl, is shown in Reaction Scheme V.

##STR00009##

Ethyl pyrazole-4-carboxylate is reacted with
1-(bromomethyl)-3-(trifluoromethyl)benzene in acetone in the presence of
potassium carbonate. The product, ethyl
1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylate, is then
hydrolyzed with potassium hydroxide in methanol, to provide
1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid.

Utility Testing and Administration

General Utility

[0184]The method and pharmaceutical compositions of the invention are
effective in the prevention of airway remodeling and/or pulmonary
inflammation in a mammal that is predisposed to airway remodeling and/or
pulmonary inflammation. The predisposition may be due to genetic
abnormalities, disease states, and/or environmental conditions that have
been shown to induce airway remodeling and/or pulmonary inflammation.

Testing

[0185]Activity testing is conducted as described in those patents and
patent applications referenced above, and in the Examples below, and by
methods apparent to one skilled in the art.

Administration

[0186]The compounds of Formula I may be administered in either single or
multiple doses by any of the accepted modes of administration of agents
having similar utilities, for example as described in those patents and
patent applications incorporated by reference, including buccal,
intranasal, intra-arterial injection, intravenously, intraperitoneally,
parenterally, intramuscularly, subcutaneously, orally, or as an inhalant.

[0187]Oral administration is the preferred route for administration of the
compounds of Formula I. Administration may be via capsule or enteric
coated tablets, or the like. In making the pharmaceutical compositions
that include at least one compound of Formula I, the active ingredient is
usually diluted by an excipient and/or enclosed within such a carrier
that can be in the form of a capsule, sachet, paper or other container.
When the excipient serves as a diluent, in can be a solid, semi-solid, or
liquid material (as above), which acts as a vehicle, carrier or medium
for the active ingredient. Thus, the compositions can be in the form of
tablets, pills, powders, lozenges, sachets, cachets, elixirs,
suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a
liquid medium), ointments containing, for example, up to 10% by weight of
the active compound, soft and hard gelatin capsules, sterile injectable
solutions, and sterile packaged powders.

[0189]The compositions of the invention can be formulated so as to provide
quick, sustained or delayed release of the active ingredient after
administration to the patient by employing procedures known in the art.
Controlled release drug delivery systems for oral administration include
osmotic pump systems and dissolutional systems containing polymer-coated
reservoirs or drug-polymer matrix formulations. Examples of controlled
release systems are given in U.S. Pat. Nos. 3,845,770; 4,326,525;
4,902514; and 5,616,345. Another formulation for use in the methods of
the present invention employs transdermal delivery devices ("patches").
Such transdermal patches may be used to provide continuous or
discontinuous infusion of the compounds of the present invention in
controlled amounts. The construction and use of transdermal patches for
the delivery of pharmaceutical agents is well known in the art. See,
e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may
be constructed for continuous, pulsatile, or on demand delivery of
pharmaceutical agents.

[0190]Adenosine A2B receptor antagonists such as the compounds of
Formula I are effective over a wide dosage range and is generally
administered in a pharmaceutically effective amount. Typically, for oral
administration, each dosage unit contains from 1 mg to 2 g of an
adenosine A2B receptor antagonist, more commonly from 1 to 700 mg,
and for parenteral administration, from 1 to 700 mg of an adenosine
A2B receptor antagonist, more commonly about 2 to 200 mg. It will be
understood, however, that the amount of the adenosine A2B receptor
antagonist actually administered will be determined by a physician, in
the light of the relevant circumstances, including the condition to be
treated, the chosen route of administration, the actual compound
administered and its relative activity, the age, weight, and response of
the individual patient, the severity of the patient's symptoms, and the
like.

[0191]For preparing solid compositions such as tablets, the principal
active ingredient is mixed with a pharmaceutical excipient to form a
solid preformulation composition containing a homogeneous mixture of a
compound of the present invention. When referring to these preformulation
compositions as homogeneous, it is meant that the active ingredient is
dispersed evenly throughout the composition so that the composition may
be readily subdivided into equally effective unit dosage forms such as
tablets, pills and capsules.

[0192]The tablets or pills of the present invention may be coated or
otherwise compounded to provide a dosage form affording the advantage of
prolonged action, or to protect from the acid conditions of the stomach.
For example, the tablet or pill can comprise an inner dosage and an outer
dosage component, the latter being in the form of an envelope over the
former. The two components can be separated by an enteric layer that
serves to resist disintegration in the stomach and permit the inner
component to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or coatings,
such materials including a number of polymeric acids and mixtures of
polymeric acids with such materials as shellac, cetyl alcohol, and
cellulose acetate.

[0193]Compositions for inhalation or insufflation include solutions and
suspensions in pharmaceutically acceptable, aqueous or organic solvents,
or mixtures thereof, and powders. The liquid or solid compositions may
contain suitable pharmaceutically acceptable excipients as described
supra. Preferably the compositions are administered by the oral or nasal
respiratory route for local or systemic effect. Compositions in
preferably pharmaceutically acceptable solvents may be nebulized by use
of inert gases. Nebulized solutions may be inhaled directly from the
nebulizing device or the nebulizing device may be attached to a face mask
tent, or intermittent positive pressure breathing machine, Solution,
suspension, or powder compositions may be administered, preferably orally
or nasally, from devices that deliver the formulation in an appropriate
manner.

[0194]The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of skill
in the art that the techniques disclosed in the examples which follow
represent techniques discovered by the inventor to function well in the
practice of the invention, and thus can be considered to constitute
preferred modes for its practice. However, those of skill in the art
should, in light of the present disclosure, appreciate that many changes
can be made in the specific embodiments which are disclosed and still
obtain a like or similar result without departing from the spirit and
scope of the invention.

Example 1

Preparation of a Compound of Formula (5)

[0195]A. Preparation of a Compound of Formula (5) in which R2 is
Ethyl

##STR00010##

[0196]A solution of sodium ethoxide was prepared from sodium (4.8 g, 226
mmol) and dry ethanol (150 ml). To this solution was added
amino-N-ethylamide (10 g, 113 in mol) and ethyl cyanoacetate (12.8 g, 113
mmol). This reaction mixture was stirred at reflux for 6 hours, cooled,
and solvent removed from the reaction mixture under reduced pressure. The
residue was dissolved in water (50 ml), and the pH adjusted to 7 with
hydrochloric acid. The mixture was allowed to stand overnight at
0° C., and the precipitate filtered off, washed with water and
air-dried, to provide 6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione, a
compound of formula (5).

[0198]Similarly, following the procedure of Example 1A, but replacing
amino-N-ethylamide with amino-N-methylamide,
6-amino-1-methyl-1,3-dihydropyrimidine-2,4-dione was prepared.

C. Preparation of a Compound of Formula (5) varying R2

[0199]Similarly, following the procedure of Example 1A, but replacing
amino-N-ethylamide with other compounds of formula (4), other compounds
of formula (5) are prepared.

Example 2

Preparation of a Compound of Formula (6)

[0200]A. Preparation of a Compound of Formula (6) in which R2 is
Ethyl

##STR00011##

[0201]A suspension of 6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione
(0.77 g, 5 mmol) in anhydrous N,N-dimethylacetamide (25 ml) and
N,N-dimethylformamide dimethylacetal (2.7 ml, 20 mmol) and was warmed at
40° C. for 90 minutes. Solvent was then removed under reduced
pressure, and the residue triturated with ethanol, filtered, and washed
with ethanol, to provide
6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-1,3-dihydropyrimidine-2,4-dione,
a compound of formula (6).

[0203]Similarly, following the procedure of Example 2A, but replacing
6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with
6-amino-1-methyl-1,3-dihydropyrimidine-2,4-dione,
6-[2-(dimethylamino)-1-azavinyl]-1-methyl-1,3-dihydropyrimidine-2,4-dione
was prepared.

C. Preparation of a Compound of Formula (6) varying R2

[0204]Similarly, following the procedure of Example 2A, but replacing
6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with other compounds of
formula (5), other compounds of formula (6) are prepared.

Example 3

Preparation of a Compound of Formula (7)

[0205]A. Preparation of a Compound of Formula (7) in which R1 is
n-Propyl and R2 is Ethyl

##STR00012##

[0206]A mixture of a solution of
6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-1,3-dihydropyrimidine-2,4-dione
(1.5 g, 7.1 mmol) in dimethylformamide (25 ml), potassium carbonate (1.5
g, 11 mmol) and n-propyl iodide (1.54 g, 11 mmol) was stirred at
80° C. for 5 hours. The reaction mixture was cooled to room
temperature, filtered, the solvents were evaporated and the product of
formula (7),
6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-propyl-1,3-dihydropyrimidine-2-
,4-dione, was used as such in the next reaction.

B. Preparation of a Compound of Formula (7), varying R1 and R2

[0207]Similarly, following the procedure of Example 3A, but replacing
6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-1,3-dihydropyrimidine-2,4-dione
with other compounds of formula (6), the following compounds of formula
(7) were prepared:
[0208]6-[2-(dimethylamino)-1-azavinyl]-1-methyl-3-propyl-1,3-dihydropyrim-
idine-2,4-dione.
[0209]6-[2-(dimethylamino)-1-azavinyl]-1-methyl-3-cyclopropylmethyl-1,3-d-
ihydropyrimidine-2,4-dione;
[0210]6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-cyclopropylmethyl-1,3-di-
hydropyrimidine-2,4-dione;
[0211]6-[2-(dimethylamino)-1-azavinyl]-1-methyl-3-(2-methylpropyl)-1,3-di-
hydropyrimidine-2,4-dione; and
[0212]6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-(2-methylpropyl)-1,3-dih-
ydropyrimidine-2,4-dione.C. Preparation of a Compound of Formula (7),
varying R1 and R2

[0213]Similarly, following the procedure of Example 3A, but replacing
6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-1,3-dihydropyrimidine-2,4-dione
with other compounds of formula (6), other compounds of formula (7) are
prepared.

Example 4

Preparation of a Compound of Formula (8)

[0214]A. Preparation of a Compound of Formula (8) in which R1 is
n-Propyl and R2 is Ethyl

##STR00013##

[0215]A solution of
6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-propyl-1,3-dihydropyrimidine-2-
,4-dione (2.1 g) was dissolved in a mixture of methanol (10 ml) and 28%
aqueous ammonia solution (20 ml), and stirred for 72 hours at room
temperature. Solvent was then removed under reduced pressure, and the
residue purified by chromatography on a silica gel column, eluting with a
mixture of dichloromethane/methanol (15/1), to provide
6-amino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compound of
formula (8).

[0217]Similarly, following the procedure of Example 4A, but replacing
6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-propyl-1,3-dihydropyrimidine-2-
,4-dione with other compounds of formula (7), the following compounds of
formula (8) were prepared:
[0218]6-amino-1-methyl-3-propyl-1,3-dihydropyrimidine-2,4-dione;
[0219]6-amino-1-methyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dion-
e; [0220]6-amino-1-ethyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dio-
ne; [0221]6-amino-1-methyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-di-
one; and [0222]6-amino-1-ethyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,-
4-dione.C. Preparation of a Compound of Formula (7) varying R1 and
R2

[0223]Similarly, following the procedure of Example 4A, but replacing
6-[2-(dimethylamino)-1-azavinyl]-1-ethyl-3-propyl-1,3-dihydropyrimidine-2-
,4-dione with other compounds of formula (7), other compounds of formula
(8) are prepared.

Example 5

Preparation of a Compound of Formula (1)

[0224]A. Preparation of a Compound of Formula (1) in which R1 is
n-Propyl and R2 is Ethyl

##STR00014##

[0225]To a solution of
6-amino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione (1.4 g, 7.1
mmol) in a mixture of 50% acetic acid/water (35 ml) was added sodium
nitrite (2 g, 28.4 mmol) in portions over a period of 10 minutes. The
mixture was stirred at 70° C. for 1 hour, then the reaction
mixture concentrated to a low volume under reduced pressure. The solid
was filtered off, and washed with water, to provide
6-amino-1-ethyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione, a
compound of formula (1).

[0226]MS m/z 227.05 (M+), 249.08 (M++Na)

B. Preparation of a Compound of Formula (1), varying R1 and R2

[0227]Similarly, following the procedure of Example 5A, but replacing
6-amino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione with other
compounds of formula (8), the following compounds of formula (1) were
prepared: [0228]6-amino-1-methyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-
-2,4-dione; [0229]6-amino-1-methyl-3-cyclopropylmethyl-5-nitro
so-1,3-dihydropyrimidine-2,4-dione;
[0230]6-amino-1-ethyl-3-cyclopropylmethyl-5-nitro
so-1,3-dihydropyrimidine-2,4-dione;
[0231]6-amino-1-methyl-3-(2-methylpropyl)-5-nitro
so-1,3-dihydropyrimidine-2,4-dione; and
[0232]6-amino-1-ethyl-3-(2-methylpropyl)-5-nitro
so-1,3-dihydropyrimidine-2,4-dione.C. Preparation of a Compound of
Formula (1) varying R1 and R2

[0233]Similarly, following the procedure of Example 5A, but replacing
6-amino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione with other
compounds of formula (8), other compounds of formula (1) are prepared.

Example 6

Preparation of a Compound of Formula (2)

[0234]A. Preparation of a Compound of Formula (2) in which R1 is
n-Propyl and R2 is Ethyl

##STR00015##

[0235]To a solution of
6-amino-1-ethyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione (300
mg) in methanol (10 ml) was added 10% palladium on carbon catalyst (50
mg), and the mixture was hydrogenated under hydrogen at 30 psi for 2
hours. The mixture was filtered through celite, and solvent was removed
from the filtrate under reduced pressure, to provide
5,6-diamino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compound
of formula (2).

[0236]MS m/z 213.03 (M+), 235.06 (M++Na)

B. Preparation of a Compound of Formula (2), varying R1 and R2

[0237]Similarly, following the procedure of Example 6A, but replacing
6-amino-1-ethyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione with
other compounds of formula (1), the following compounds of formula (2)
were prepared:
[0238]5,6-diamino-1-methyl-3-propyl-1,3-dihydropyrimidine-2,4-dione;
[0239]5,6-diamino-1-methyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4--
dione; [0240]5,6-diamino-1-ethyl-3-cyclopropylmethyl-1,3-dihydropyrimidine-
-2,4-dione;
[0241]5,6-amino-1-methyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dio-
ne; and [0242]5,6-diamino-1-ethyl-3-(2-methylpropyl)-1,3-dihydropyrimidine-
-2,4-dione.C. Preparation of a Compound of Formula (2) varying R1 and
R2

[0243]Similarly, following the procedure of Example 6A, but replacing
6-amino-1-ethyl-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione with
other compounds of formula (1), other compounds of formula (2) are
prepared.

Example 7

Preparation of a Compound of Formula (3)

[0244]A. Preparation of a Compound of Formula (3) in which R1 is
n-Propyl, R2 is Ethyl, X is 1,4-Pyrazolyl, Y is Methylene, and Z is
3-Trifluoromethylphenyl

[0264]Similarly, following the procedure of Example 7A, but optionally
replacing 5,6-diamino-1-ethyl-3-propyl-1,3-dihydropyrimidine-2,4-dione
with other compounds of formula (2), and optionally replacing
1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid with
other compounds of formula Z--Y--X--CO2H, other compounds of formula
(3) are prepared.

Example 8

Preparation of a Compound of Formula I

[0265]A. Preparation of a Compound of Formula I in which R1 is
n-Propyl, R2 is Ethyl, X is 1,4-Pyrazolyl, Y is Methylene, and Z is
3-Trifluoromethylphenyl

##STR00017##

[0266]A mixture of
N-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(-
trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamide (80 mg, 0.17 mmol),
10% aqueous sodium hydroxide (5 ml), and methanol (5 ml) was stirred at
100° C. for 2 hours. The mixture was cooled, methanol removed
under reduced pressure, and the residue diluted with water and acidified
with hydrochloric acid. The precipitate was filtered off, washed with
water, then methanol, to provide
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,-
3,7-trihydropurine-2,6-dione, a compound of Formula I.

[0284]Similarly, following the procedure of Example 8A, but replacing
N-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(-
trifluoromethyl)phenyl]-methyl}pyrazol-3-yl)carboxamide with other
compounds of formula (3), other compounds of Formula I are prepared.

Example 9

Preparation of a Compound of Formula (10)

[0285]A. Preparation of a Compound of Formula (10) in which R1 is
n-Propyl

##STR00018##

[0286]A mixture of 6-aminouracil (5.08 g, 40 mmol), hexamethyldisilazane
(50 ml), and ammonium sulfate (260 mg, 1.96 mmol) was refluxed for 12
hours. After cooling, the solid was filtered off, and solvent was removed
from the filtrate under reduced pressure to provide the
trimethylsilylated derivative of 6-aminouracil.

[0287]The product was dissolved in toluene (1.5 ml), and iodopropane (7.8
ml, 80 mmol) and heated in an oil bath at 120° C. for 2 hours. The
reaction mixture was then cooled to 0° C., and saturated aqueous
sodium bicarbonate added slowly. The resulting precipitate was filtered
off, and washed sequentially with water, toluene, and ether, to provide
6-amino-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compound of formula
(10), which was used in the next reaction with no further purification.

[0289]Similarly, following the procedure of Example 9A, but replacing
iodopropane with other alkyl halides of formula R1Hal, other
compounds of formula (10) are prepared, including:
[0290]6-amino-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione; and
[0291]6-amino-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione.

Example 10

Preparation of a Compound of Formula (11)

[0292]A. Preparation of a Compound of Formula (10) in which R1 is
n-Propyl

##STR00019##

[0293]To a solution of 6-amino-3-propyl-1,3-dihydropyrimidine-2,4-dione
(5.6 g) in a mixture of 50% acetic acid/water (160 ml) at 70° C.
was added sodium nitrite (4.5 g) in portions over a period of 15 minutes.
The mixture was stirred at 70° C. for 45 minutes, then the
reaction mixture concentrated to a low volume under reduced pressure. The
solid was filtered off, and washed with water, to provide
6-amino-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compound of
formula (11).

[0295]Similarly, following the procedure of Example 10A, but replacing
6-amino-3-propyl-1,3-dihydropyrimidine-2,4-dione with other compounds of
formula (10), other compounds of formula (11) are prepared, including:
[0296]6-amino-5-nitroso-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dio-
ne; and [0297]6-amino-5-nitroso-3-(2-methylpropyl)-1,3-dihydropyrimidine-2-
,4-dione.

Example 11

Preparation of a Compound of Formula (12)

[0298]A. Preparation of a Compound of Formula (12) in which R1 is
n-Propyl

##STR00020##

[0299]To a solution of
6-amino-5-nitroso-3-propyl-1,3-dihydropyrimidine-2,4-dione (5.4 g, 27
mmol) in 12.5% aqueous ammonia (135 ml) at 70° C. was added sodium
dithionite (Na2S2O4, 9.45 g, 54 mmol) in portions over 15
minutes, and the mixture was stirred for 20 minutes. The solution was
concentrated under reduced pressure, cooled to 5° C., the
precipitate filtered off, and washed with cold water, to provide
5,6-diamino-3-propyl-1,3-dihydropyrimidine-2,4-dione, a compound of
formula (12).

[0301]Similarly, following the procedure of Example 11A, but replacing
6-amino-3-propyl-1,3-dihydropyrimidine-2,4-dione with other compounds of
formula (11), other compounds of formula (12) are prepared, including:
[0302]5,6-diamino-3-cyclopropylmethyl-1,3-dihydropyrimidine-2,4-dione;
and [0303]5,6-diamino-3-(2-methylpropyl)-1,3-dihydropyrimidine-2,4-dione.

Example 12

Preparation of a Compound of Formula (13)

[0304]A. Preparation of a Compound of Formula (13) in which R1 is
n-Propyl, X is 1,4-Pyrazolyl, Y is Methylene, and Z is
3-Trifluoromethylphenyl

##STR00021##

[0305]To a mixture of 5,6-diamino-3-propyl-1,3-dihydropyrimidine-2,4-dione
(2.3 g, 126 mmol) and
1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid (3.79 g,
14 mmol) in methanol (50 ml) was added
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.67 g, 14
mmol), and the reaction mixture was stirred for 3 days at room
temperature (although less time is acceptable). The precipitate was
filtered off, and was washed sequentially with water, and methanol. The
product was dried under vacuum to provide
N-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluor-
omethyl)phenyl]methyl}pyrazol-4-yl)carboxamide, a compound of formula
(13).

B. Alternative Preparation of a Compound of Formula (3) in which R1
is n-Propyl, X is 1,4-Pyrazolyl, Y is Methylene, and Z is
3-Trifluoromethylphenyl

[0307]A solution of
1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid (1 g, 3.7
mmol) in thionyl chloride (1 ml) was heated at 70° C. for 4 hours.
Excess thionyl chloride was distilled off, and the residue treated with
methylene chloride/hexanes. The solvent was removed under reduced
pressure, and the residue dissolved in acetonitrile. This solution was
added to a suspension of
5,6-diamino-3-propyl-1,3-dihydropyrimidine-2,4-dione (2.3 g, 126 mmol)
and triethylamine (1 ml) in acetonitrile (20 ml) at 0° C., and
stirred for 16 hours. The reaction mixture was quenched with water (5
ml), acidified with hydrochloric acid, stirred for 30 minutes, and the
precipitate filtered off. The product was washed with ether, to provide
N-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluor-
omethyl)phenyl]methyl}pyrazol-4-yl)carboxamide, a compound of formula
(13).

[0326]A. Preparation of a Compound of Formula (3) in which R1 is
n-Propyl, R2 is Ethyl, X is 1,4-Pyrazolyl, Y is Methylene, and Z is
3-Trifluoromethylphenyl

##STR00022##

[0327]A mixture of a solution of
N-(6-amino-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluor-
omethyl)-phenyl]methyl}pyrazol-3-yl)carboxamide (872 mg, 2 mmol) in
dimethylformamide (10 ml), potassium carbonate (552 mg, 4 mmol) and ethyl
iodide (0.24 ml, 3 mmol) was stirred at room temperature overnight. The
reaction mixture was filtered, and the solvent was evaporated from the
filtrate under reduced pressure. The residue was stirred with water for
two hours at room temperature, and the precipitate filtered off, washed
with water, and then dissolved in methanol. The solvent was then removed
under reduced pressure to provide
N-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(-
trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide, a compound of
formula (3).

[0349]Similarly, following the procedure of Example 14A, but replacing
N-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(-
trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamide with other
compounds of formula (13), other compounds of Formula I are prepared,
including those listed in Example 8.

Example 15

Preparation of a Compound of Formula (14)

[0350]A. Preparation of a Compound of Formula (14) in which R2 is
Ethyl

##STR00024##

[0351]To a solution of 6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione
(5.0 g, 32.3 mmol) in a mixture of 50% acetic acid/water (50 ml) at
70° C. was added sodium nitrite (4.45 g, 64.5 mmol) in portions
over a period of 30 minutes. The mixture was stirred at 70° C. for
a further 30 minutes. The reaction mixture was cooled, and the
precipitate filtered off, and washed with water, then methanol, to
provide 6-amino-1-ethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione, a
compound of formula (14).

[0353]Similarly, following the procedure of Example 15A, but replacing
6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with
6-amino-1-methyl-1,3-dihydropyrimidine-2,4-dione,
6-amino-1-methyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione was prepared.

C. Preparation of a Compound of Formula (14), varying R2

[0354]Similarly, following the procedure of Example 15A, but replacing
6-amino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with other compounds of
formula (5), other compounds of formula (14) are prepared.

Example 16

Preparation of a Compound of Formula (15)

[0355]A. Preparation of a Compound of Formula (15) in which R2 is
Ethyl

##STR00025##

[0356]To a solution of
6-amino-1-ethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione (3.9 g, 21.2
mmol) in 14.5% aqueous ammonia (50 ml) at 50° C. was added sodium
dithionite (Na2S2O4, 7.37 g, 42.4 mmol) in portions over
15 minutes, and the mixture was stirred for 20 minutes. The solution was
concentrated under reduced pressure to a volume of 30 ml, cooled to
5° C., the precipitate filtered off, and washed with cold water,
to provide 5,6-diamino-1-ethyl-1,3-dihydropyrimidine-2,4-dione, a
compound of formula (15).

[0358]Similarly, following the procedure of Example 16A, but replacing
6-amino-1-ethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione with
6-amino-1-methyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione,
5,6-diamino-1-methyl-1,3-dihydropyrimidine-2,4-dione was prepared.

C. Preparation of a Compound of Formula (15), varying R2

[0359]Similarly, following the procedure of Example 16A, but replacing
6-amino-1-ethyl-5-nitroso-1,3-dihydropyrimidine-2,4-dione with other
compounds of formula (14), other compounds of formula (15) are prepared.

Example 17

Preparation of a Compound of Formula (16)

[0360]A. Preparation of a Compound of Formula (16) in which R2 is
Ethyl, X is 1,4-Pyrazolyl, Y is Methylene, and Z is
3-Trifluoromethylphenyl

##STR00026##

[0361]To a mixture of 5,6-diamino-1-ethyl-1,3-dihydropyrimidine-2,4-dione
(2 g, 11.76 mmol) and
1-{[3-(trifluoromethyl)phenyl]methyl}pyrazole-4-carboxylic acid (3.5 g,
12.94 mmol) in methanol (50 ml) was added
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.47 g,
12.94 mmol), and the reaction mixture was stirred for 16 hours at room
temperature. Solvent was removed under reduced pressure, and the residue
was washed with water and methanol. The product was dried under vacuum to
provide N-(6-amino-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3-(t-
rifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide, a compound of
formula (16).

[0363]Similarly, following the procedure of Example 17A, but replacing
5,6-diamino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with
5,6-diamino-1-methyl-1,3-dihydropyrimidine-2,4-dione,
N-(6-amino-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluor-
omethyl)phenyl]methyl}pyrazol-4-yl)carboxamide was prepared.

C. Preparation of a Compound of Formula (16), varying R2, X, Y, and Z

[0364]Similarly, following the procedure of Example 16A, but replacing
5,6-diamino-1-ethyl-1,3-dihydropyrimidine-2,4-dione with other compounds
of formula (14), other compounds of formula (15) are prepared.

Example 18

Preparation of a Compound of Formula (3)

[0365]A. Preparation of a Compound of Formula (3) in which R1 is
n-Propyl, R2 is Ethyl, X is 1,4-Pyrazolyl, Y is Methylene, and Z is
3-Trifluoromethylphenyl

##STR00027##

[0366]A mixture of a solution of
N-(6-amino-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoro-
methyl)phenyl]methyl}pyrazol-3-yl)carboxamide (1.5 g, 3.55 mmol) in
dimethylformamide (30 ml), potassium carbonate (980 mg, 7.1 mmol) and
propyl iodide (724 mg, 4.26 mmol) was stirred at room temperature
overnight. Water was added, and the precipitate filtered off, to provide
N-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(-
trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide, a compound of
formula (3), which was used in the next reaction with no further
purification.

[0368]Similarly, following the procedure of Example 18A, but replacing
N-(6-amino-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3-(trifluoro-
methyl)phenyl]-methyl}pyrazol-3-yl)carboxamide with
N-(6-amino-1-methyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl)),
N-(6-amino-1-methyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3--
(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)carboxamide was prepared.

[0369]Similarly, following the procedure of Example 18A, but optionally
replacing N-(6-amino-1-ethyl-2,4-dioxo(1,3-dihydropyrimidin-5-yl))(1-{[3--
(trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamide with other
compounds of formula (15), and optionally replacing propyl iodide with
other compounds of formula R1Hal, other compounds of formula (3) are
prepared.

Example 19

Preparation of a Compound of Formula

[0370]A. Preparation of a Compound of Formula 1 in which R1 is
n-Propyl, R2 is Ethyl, X is 1,4-Pyrazolyl, Y is Methylene, and Z is
3-Trifluoromethylphenyl

##STR00028##

[0371]A mixture of
N-(6-amino-1-ethyl-2,4-dioxo-3-propyl(1,3-dihydropyrimidin-5-yl))(1-{[3-(-
trifluoromethyl)phenyl]methyl}pyrazol-3-yl)carboxamide (300 mg, 464 mmol),
20% aqueous sodium hydroxide (5 ml), and methanol (10 ml) was stirred at
80° C. for 3 hours. The mixture was cooled, methanol removed under
reduced pressure, and the remaining mixture was acidified with
hydrochloric acid to pH 2. The precipitate was filtered off, washed with
water and methanol, to provide
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}pyrazol-4-yl)-1,-
3,7-trihydropurine-2,6-dione, a compound of Formula I.

[0373]Human A2B adenosine receptor cDNA was stably transfected into
HEK-293 cells (referred to as HEK-A2B cells). Monolayers of HEK-A2B cells
were washed with PBS once and harvested in a buffer containing 10 mM
HEPES (pH 7.4), 10 mM EDTA and protease inhibitors. These cells were
homogenized in polytron for 1 minute at setting 4 and centrifuged at
29000 g for 15 minutes at 4° C. The cell pellets were washed once
with a buffer containing 10 mM HEPES (pH7.4), 1 mM EDTA and protease
inhibitors, and were resuspended in the same buffer supplemented with 10%
sucrose. Frozen aliquots were kept at -80° C.

[0375]Human A1, A2A, A3 adenosine receptor cDNAs were
stably transfected into either CHO or HEK-293 cells (referred to as
CHO-A1, HEK-A2A, CHO-A3). Membranes were prepared from
these cells using the same protocol as described above. Competition
assays were started by mixing 0.5 nM 3H-CPX (for CHO-A1), 2 nM
3H-ZM214385 (HEK-A2A) or 0.1 nM 125I-AB-MECA (CHO-A3)
with various concentrations of test compounds and the perspective
membranes in TE buffer (50 mM Tris and 1 mM EDTA of CHO-A1 and
HEK-A2A) or TEM buffer (50 mM. Tris, 1 mM EDTA and 10 mM MgCl2
for CHO-A3) supplemented with 1 Unit/mL adenosine deaminase. The
assays were incubated for 90 minutes, stopped by filtration using Packard
Harvester and washed four times with ice-cold TM buffer (10 mM Tris, 1 mM
MgCl2, pH 7.4). Non specific binding was determined in the presence
of 1 μM CPX (CHO-A1), 1 μM ZM241385 (HEK-A2A) and 1 μM
IB-MECA (CHO-A3). The affinities of compounds (I.e. Ki values) were
calculated using GraphPad® software.

cAMP Measurements

[0376]Monolayer of transfected cells were collected in PBS containing 5 mM
EDTA. Cells were washed once with DMEM and resuspended in DMEM containing
1 Unit/mL adenosine deaminase at a density of 100,000-500,000 cells/ml.
100 μl of the cell suspension was mixed with 25 μl containing
various agonists and/or antagonists and the reaction was kept at
37° C. for 15 minutes. At the end of 15 minutes, 125 μl 0.2N
HCl was added to stop the reaction. Cells were centrifuged for 10 minutes
at 1000 rpm. 100 μl of the supernatant was removed and acetylated. The
concentrations of cAMP in the supernatants were measured using the direct
cAMP assay from Assay Design. A2A and A2B adenosine receptors
are coupled to Gs proteins and thus agonists for A2A adenosine
receptor (such as CGS21680) or for A2A adenosine receptor (such as
NECA) increase the cAMP accumulations whereas the antagonists to these
receptors prevent the increase in cAMP accumulations-induced by the
agonists. A1 and A3 adenosine receptors are coupled to Gi
proteins and thus agonists for A1 adenosine receptor (such as CPA)
or for A3 adenosine receptor (such as IB-MECA) inhibit the increase
in cAMP accumulations-induced by forskolin. Antagonists to A1 and
A3 receptors prevent the inhibition in cAMP accumulations.

Example 21

Effect of A2B Antagonist on Airway Restructuring in Mouse Model

[0377]The model system being used is the adenosine deaminase
(ADA)-deficient mouse model of adenosine-dependent pulmonary injury. In
this model, elevations in adenosine are associated with increased
pulmonary inflammation and airway remodeling. Many of the features seen
in these mice resemble those observed in patients with various forms of
chronic lung disease including severe asthma, COPD and pulmonary
fibrosis. The approach is to treat these mice with the A2B AR
antagonist
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyrazol--
4-yl))-1,3,7-trihydropurine-2,6-dione as a means to probe A2B AR
contributions to pulmonary inflammation and injury in ADA-deficient mice,
which should provide insight into the efficacy of this drug for the
treatment of chronic lung diseases.

[0378]Mice treated by intraperitoneal (ip) injection twice daily with 1
mg/kg 3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropy-
razol-4-yl))-1,3,7-trihydropurine-2,6-dione. The specific protocol is as
follows: ADA-deficient (ADA-/-) or ADA containing (ADA+) mice were
identified at birth by screening of ADA enzymatic activity in the blood.
ADA-/- mice were maintained on ADA enzyme therapy from postnatal day 2
until postnatal day 21. At this stage, treatments with
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyrazol--
4-yl))-1,3,7-trihydropurine-2,6-dione or vehicle controls (corn
oil/ethanol/DMSO) were initiated. Treatments consisted of an ip injection
in the morning (8-9 AM) and in the evening (5-6 pm). Treatments were
given daily for 17 days, and the experiment was terminated on postnatal
day 38. Treatment groups included ADA-/- or ADA+ mice receiving
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyrazol--
4-yl))-1,3,7-trihydropurine-2,6-dione, vehicle, or no treatment. All mice
were littermates and were therefore strain matched. Both males and
females were included in these experiments. At the termination of the
experiment, serum was collected for pharmacokinetics (PK) analysis of
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyrazol--
4-yl))-1,3,7-trihydropurine-2,6-dione levels and various pulmonary
endpoints were examined.

[0379]Results from these studies indicate that treatment of ADA-/- mice
with 3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyr-
azol-4-yl))-1,3,7-trihydropurine-2,6-dione resulted in a significant
reduction in pulmonary inflammation and airway destruction. Perhaps the
most dramatic observation from this study was the general health of the
animals at the end of the treatment protocol. ADA-/- mice either treated
with vehicle or untreated exhibited outward signs of severe respiratory
distress that included rapid breathing and hunched posture. These
features were not observed in ADA-/- mice treated with
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyrazol--
4-yl))-1,3,7-trihydropurine-2,6-dione.

Methods:

Mice

[0380]ADA-deficient mice were generated and genotyped as described
(Blackburn et al. (1998) J Biol Chem 273:5093-5100 and Young et al.
(2004) J. Immunol. 173:1380-1389. Mice homozygous for the null Ada allele
were designated ADA-deficient (ADA.sup.-/-), while mice heterozygous for
the null Ada allele were designated as ADA control mice (ADA+). All
mice were on a mixed 129sv/C57BL/6J background and all phenotypic
comparisons were performed amongst littermates. Animal care was in
accordance with institutional and NIH guidelines. Mice were housed in
ventilated cages equipped with microisolator lids and maintained under
strict containment protocols. No evidence of bacterial, parasitic, or
fungal infection was found, and serologies on cage littermates were
negative for 12 of the most common murine viruses.

Cellular Differentials and Histology

[0381]Mice were anesthetized with avertin, and lungs were lavaged four
times with 0.3 ml PBS, and 0.95-1 ml of pooled lavage fluid was
recovered. Total cell counts were determined using a hemocytometer, and
aliquots were cytospun onto microscope slides and stained with Diff-Quick
(Dade Nehring) for cellular differentials. Lungs were then infused with
4% paraformaldehyde in PBS at 25 cm of pressure and then fixed overnight
at 4° C. Fixed lung samples were rinsed in PBS, dehydrated, and
embedded in paraffin. Sections (5 μm) were collected on microscope
slides and stained with hematoxylin and eosin (H&E; Shandon-Lipshaw) or
Masson's trichrome (EM Science), according to manufacturer's
instructions.

Analysis of mRNA

[0382]Mice were anesthetized and the lungs were rapidly removed and frozen
in liquid nitrogen. RNA was isolated from frozen lung tissue using TRIzol
Reagent (Life Technologies Inc., Grand Island, N.Y., USA). RNA samples
were then DNase treated and subjected to quantitative real-time RT-PCR.
The primers, probes and procedures for real-time RT-PCR were described
previously in Sun et al. (2005) J Clin Invest 115:35-43. Reactions were
carried out on a Smart Cycler rapid thermal cycler system (Cepheid,
Sunnyvale, Calif.). Specific transcript levels were determined using
Smart Cycler analysis software through comparison to a standard curve
generated from the PCR amplification of template dilutions.

Collagen Quantification

[0383]The Sircol collagen assay (Biocolor Ltd., Belfast N. Ireland) was
performed on snap frozen whole lungs. Lungs were homogenized in 5 ml.
0.5M. Acetic acid with 20 mg of pepsin and incubated with shaking for 24
hrs at 4° C. Homogenate was spun at 4000 rpm and supernatant was
assayed for pepsin soluble collagen according to manufacture's
instructions.

α-SMA and TGF-β1 Immunohistochemistry

[0384]Immunohistochemistry was performed on 5 μm sections cut from
formalin-fixed, paraffin embedded lungs. Sections were rehydrated through
graded ethanols to water, endogenous peroxidases were quenched with 3%
hydrogen peroxide, antigen retrieval was performed (DAKO Corp.,
Carpenteria, Calif.), and endogenous avidin and biotin was blocked with
the Biotin Blocking System (DAKO Corp.). For a-smooth muscle actin (sma)
staining, slides were processed with the Mouse on Mouse Kit, and the ABC
Elite Streptavidin Reagents (Vector Laboratories, Burlingame, Calif.) and
incubated with a 1:500 dilution of a a-sma monoclonal antibody (Sigma,
monoclonal clonel A-4) overnight at 4° C. Sections were developed
with DAB (Sigma) and counterstained with Methyl Green.

Determination of Alveolar Airspace Size

[0385]The size of alveolar airways was determined in pressure infused
lungs by measuring mean chord lengths on H&E-stained lung sections
(Blackburn et al. (2000) J Exp Med 192:159-170). Representative images
were digitized, and a grid consisting of 53 black lines at 10.5-μm
intervals was overlaid on the image. This line grid was subtracted from
the lung images using Image-Pro® Plus (Media Cybernetics) image
analysis software, and the resultant lines were measured and averaged to
give the mean chord length of the alveolar airways. The final mean chord
lengths represent averages from 10 non-overlapping images of each lung
specimen. All quantitative studies were performed blinded with regards to
animal genotype.

Results

Histological Analysis

[0386]Histological analysis of lungs are shown in FIG. 1. The lungs of
ADA-/- mice treated with vehicle exhibited significant alveolar airway
simplification (FIG. 1B) and increased pulmonary inflammation that
consisted predominantly of accumulation of activated macrophages in the
distal airways (FIG. 1E). However, peribronchial/perivascular
inflammation was also evident (not shown). Alveolar airway simplification
and pulmonary inflammation was not evident in ADA+ vehicle treated (FIGS.
1A and D) or
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyrazol--
4-yl))-1,3,7-trihydropurine-2,6-dione or untreated mice (not shown).
Treatment of ADA-/- mice with the adenosine A2B receptor antagonist
resulted in a significant decrease in alveolar airway simplification
(FIG. 1C) and pulmonary inflammation (FIG. 1F). These findings indicate
that treatment with an A2B adenosine receptor antagonist can prevent
the development of pulmonary inflammation and airway destruction in
ADA-/- mice.

Bronchiolalveolar Lavage Analysis

[0387]Bronchiolalveolar lavage (BAL) was performed and airway cell counts
and differentials were determined (FIG. 2). Results revealed that there
was a significant reduction in the number of total cells recovered from
BAL of ADA-/- mice treated with the A2B adenosine receptor
antagonist as compared to vehicle treated ADA-/- mice (FIG. 2A). Analysis
of cellular differentials from BALs revealed a reduction in all cell
types examined including lymphocytes, neutrophils, eosinophils (FIG. 2C)
and alveolar macrophages (FIG. 2B) in ADA-/- mice treated with the
A2B adenosine receptor antagonist.

[0388]Examination of alveolar macrophages within the lungs of ADA-/- mice
with and without A2B adenosine receptor antagonist treatment
indicate that there was a difference in the degree of alveolar macrophage
activation (compare FIGS. 1E and F). These observations were confirmed by
quantifying the number of activated macrophages recovered from the BAL
(FIG. 2C). In addition, reduction in alveolar macrophage activation can
be appreciated by directly examining BAL cells cytospun onto microscope
slides (FIG. 3). These data demonstrate that there is a significant
decrease in airway inflammation in ADA-/- mice treated with an A2B
adenosine receptor antagonist showing that A2BAR antagonism can
prevent airway inflammation in mammals that are genetically and/or
environmentally predisposed to airway remodeling.

Effect on Inflammatory Markers

[0389]The ability of adenosine A2B antagonist treatment to dampen the
degree of pulmonary inflammation in ADA.sup.-/- mice prompted the
examination of the levels of key cytokines and chemokines. Whole lung RNA
extracts from ADA+ and ADA.sup.-/- mice treated with vehicle or an
A2B adenosine receptor antagonist were analyzed. IL-5, IL-4,
TNFα, RANTES and various monocyte chemoatraetant proteins (MCPs)
were found to be elevated in the lungs of ADA.sup.-/- mice treated with
vehicle; however their levels did not change with A2B adenosine
receptor antagonist treatment. In contrast, IL-6, Eotaxin I and TARC were
elevated in vehicle treated ADA.sup.-/- mice and their levels were
significantly lower in ADA.sup.-/- mice treated with the A2B
adenosine receptor antagonist. As shown in FIG. 4, these findings
demonstrate that A2BAR antagonism in ADA.sup.-/- mice is able to
prevent the expression of certain but not all pro-inflammatory cytokines
and chemokines.

The Effect on Myofibroblast Accumulation

[0390]Previous studies have demonstrated that ADA.sup.-/- mice develop
pulmonary fibrosis in conjunction with adenosine elevations. To determine
the effect of an adenosine A2B receptor antagonist treatment on
pulmonary fibrosis in ADA.sup.-/- mice the status of pulmonary
myofibroblasts were examined by staining for α-smooth muscle actin
(α-sma) (FIG. 5). No α-sma positive cells were seen in the
distal airways of ADA+ vehicle treated mice (FIG. 5A), whereas
α-sma staining was prominent in the distal airways of vehicle
treated ADA.sup.-/- mice (FIG. 5B). Few to no α-sma positive cells
were seen in the distal airways of ADA.sup.-/- mice treated with an
adenosine A2B receptor antagonist (FIG. 5C), suggesting A2BAR
antagonism can prevent the accumulation of myofibroblasts in the lung of
ADA.sup.-/- mice.

Effects on Collagen Deposition

[0391]Previous work in the ADA-/- model has demonstrated that ADA-/- mice
develop severe pulmonary fibrosis in conjunction with adenosine
elevations. To determine the effect of A2B adenosine receptor
antagonist treatment on fibrosis in ADA-/- mice we examined the degree of
collagen deposition using Mason's trichrome staining (FIG. 6).
Examination of collagen deposition revealed that there was little to no
collagen deposition in the lungs of ADA+ vehicle or adenosine
A2B receptor antagonist treated mice (FIGS. 6A and B), whereas
distal airway collagen deposition was prominent in vehicle treated ADZ
mice (FIG. 6C). Treatment of ADA.sup.-/- mice with the adenosine A2B
receptor antagonist
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyrazol--
4-yl))-1,3,7-trihydropurine-2,6-dione resulted in a reduction in collagen
deposition in the airways (FIG. 6D). Collagen production was measured by
quantifying whole lung α1-procollagen transcript levels (FIG. 6E)
and collagen protein levels in BAL fluid (FIG. 6F). Significant increases
in collagen production were seen in the lungs of ADA.sup.-/- mice treated
with vehicle, and these increases were largely prevented by CVT-6883
treatment. These findings demonstrate that adenosine A2B receptor
antagonists can prevent the development of fibrosis in ADA.sup.-/- mice
and implicate A2BAR signaling in the regulation of pulmonary
fibrosis.

Reduction in Profibrotic Mediators

[0392]TGF-β1, osteopontin (OPN) and plasminogen activator inhibitor-1
(PAI-1) are pro-fibrotic mediators that have been shown to be elevated in
the lungs of ADA.sup.-/- mice (Chunn et al (2005) J Immunol
175:1937-1946). The levels of these fibrotic regulators were elevated in
the lungs of ADA.sup.-/- mice treated with vehicle, while adenosine
A2B receptor antagonist treatment decreased expression of these
molecules (FIG. 7). These findings suggest that A2BAR antagonism can
prevent the expression of key regulators of fibrosis in the lungs of
ADA.sup.-/- mice.

Normalization of Protease/Anti-Protease Levels

[0393]Increased levels of metalloproteinases (MMPs) and inhibitors of
proteases are features of distal airway enlargement in many models
including ADA.sup.-/- mice (Sun et al. (2005) J Clin Invest 115:35-43,
Hautamaki et al. (1997) Science 277:2002-2004, Lanone et al. (2002) J
Clin Invest 110:463-474). Examination of anti-proteases and proteases in
the lungs of ADA.sup.-/- mice treated with vehicle demonstrated an
increase in the expression of TIMP-1, MMP-9 and MMP-12 (FIG. 8).
Treatment of ADA.sup.-/- mice with an adenosine A2B receptor
antagonist led to diminished expression of all three of these regulators
of alveolar integrity, suggesting that A2BAR signaling is involved
in regulating adenosine-induced protease and anti-protease expression in
the lungs of ADA.sup.-/- mice.

Effects on Alveolar Destruction

[0394]ADA-/- mice develop features of distal airway enlargement
characteristic of emphysema that are mediated by elevations in lung
adenosine levels. To examine the effect of
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyrazol--
4-yl))-1,3,7-trihydropurine-2,6-dione on the distal airway enlargement
seen in ADA-/- mice, alveolar destruction was assessed histologically and
by determining mean cord length size of the distal airways (FIG. 9). The
airways of ADA+ animals were well ordered and small when viewed
histologically (FIG. 9A). ADA-/- airways were enlarged (FIG. 9B) and
treatment of ADA-/- mice with
3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hydropyrazol--
4-yl))-1,3,7-trihydropurine-2,6-dione prevented this enlargement (FIG.
9C). Quantification of distal airway size agreed with the histological
observations (FIG. 9D). These data demonstrate that treatment of ADA-/-
mice with 3-ethyl-1-propyl-8-(1-{[3-(trifluoromethyl)phenyl]methyl}(4-hyd-
ropyrazol-4-yl))-1,3,7-trihydropurine-2,6-dione can prevent the alveolar
airway destruction seen in ADA-/- mice

Patent applications by Dewan Zeng, Palo Alto, CA US

Patent applications by Luiz Belardinelli, Palo Alto, CA US

Patent applications by Michael R. Blackburn, Pearland, TX US

Patent applications by Gilead Palo Alto, Inc.

Patent applications in class The additional hetero ring is six-membered consisting of one nitrogen and five carbons

Patent applications in all subclasses The additional hetero ring is six-membered consisting of one nitrogen and five carbons